Studies in humans and macaques have demonstrated that CD8+ T cells responses are associated with the initial control of HIV or SIV replication. Natural killer (NK) cells also influence viral control and survival. These antiviral activities are dependent on major histocompatibility complex (MHC) molecules and specific MHC genotypes have been associated with lower viral loads and slower disease progression. However, the exact correlates of protection remain unidentified and the immune responses required for an effective vaccine need to be defined. Crucial information can be obtained from experimental studies such as the infection of Asian macaques with SIV or SHIV viruses, but also from studies of African natural hosts of SIVs, which exhibit high viral loads but remain disease-free. Comparison of immune responses induced in these natural and experimental hosts infected with the same SIV strains would shed light on the protective mechanisms used by African primates to resist the development of immunodeficiency. Three macaque species are used to mimic HIV infection in pathogenesis and vaccine studies: rhesus macaques (Macaca mulatta), pig-tailed macaques (M. nemestrina) and cynomolgus monkeys (M. fascicularis). Pig-tailed macaques possess unique susceptibility and disease development characteristics that make this species particularly informative for AIDS research (high level of celllular activation, rapid disease development, suceptibility to SIVagm strains). Specific MHC class I alleles have been associated with slower disease progression and lower viral loads in humans and rhesus macaques. A similar link between MHC genetic background and disease course has not been established for pig-tailed macaques. Our research is focused on exploring how the host genetic background of macaques affects their innate and adaptive immune response to SIV and SHIV infection. Specifically, our research this year concentrated on characterizing NK cell capacity to detect infected cells. Previously, we have shown that CD8+ T cells from several SHIV-infected pig-tailed macaques responded to a SIV p27 Gag epitope (DI9 DHQAAMQII) by producing IFNg and TNFa. These animals controlled viral replication soon after inoculation and remained asymptomatic for up to 6 years with undetectable levels of plasma viral RNA. In contrast, other pig-tailed macaques inoculated under similar conditions experienced a complete and irreversible elimination of their CD4+ T cells and were euthanized within 6 months of infection. Further analyses demonstrated that CD8+ T cells responses specific to SIV Gag DI9 were restricted by the Mane-A1*082 MHC allele, potentially explaining differences in disease progression between the two groups of monkeys. However, not all infected pig-tailed macaques carrying the A1*082 allele had SIV Gag DI9 specific CD8+ T cells, as detected by intracellular IFNg staining. We performed tetramer staining to assess if this lack of detection was due to the absence of SIV Gag DI9 specific CD8+ T lymphocytes, or if such CD8+ T cells were present but remained undetected because their response was skewed toward expression of other effector molecules. Mane-A1*082 tetramers loaded with peptides SIV Gag DI9 or HI8 (a truncated peptide) were generated in collaboration with Dr David Price (Cardiff University). The presence of epitope specific CD8+ T cells was confirmed by staining CD8+ T cells with both tetramers in macaques responsive to SIV Gag DI9. Staining with both A1*082 tetramers confirmed the absence of CD8+ T cells able to recognize the epitope in unresponsive animals. However, a significant subpopulation of NK cells (6 to 22%) was labeled with both tetramers. Further analyses with a larger cohort of macaques showed that the presence of tetramer positive NK cell subsets was independent of MHC genotype or infection status. All animals with tetramer positive NK cells had a background level of staining on their CD8+ T cells. Using a third tetramer (Mane-A4*1402 molecule loaded with SIV Gag AF9 peptide), we identified three patterns of NK cell reactivity, suggesting the presence of at least three distinct NK cell receptors specific for peptide-MHC complexes. The type I receptor recognized both A1*082 tetramers whereas the type II receptor recognized only the A1*082 HI8 tetramer. The type III receptor appeared specific for A4*1402 AF9 tetramer only. To investigate the nature of these receptors, we performed phenotypic analyses of macaque NK cells positive or negative with A1*082 or A4*1402 tetramers. In all animals tested, almost all NK cells expressed the NK receptors NKG2A, NKG2D, NKp30, NKp46, the adhesion molecule CD2, the IL-2Rb chain, and the FcGRIII receptor. NK cells expressed also CD161 and KIR2D molecules but with more variation between animals. In contrast, CD56, an adhesion molecule found on all human NK cells, was only present in a very small subset of macaque NK cells. None of these markers appeared to correlate with tetramer binding patterns. The tetramer positive NK cells were observed in a limited number of animals, restricted to a minor but significant NK cell subsets, and were specific to MHC allele / peptide complexes. We hypothesized that these characteristics could result from the presence of polymorphic NK receptors involved in self, non-self and altered self-recognition. Primates express such NK cell surface molecules as a family of receptors named killer cell immunoglobulin (Ig) -like receptors (KIR). KIRs comprise activating and inhibitory molecules that recognized specific MHC allotypes in a peptide dependant manner. KIRs are encoded by a multigenic family and their population-level diversity is generated by both gene polymorphism and the number of genes carried in the genome. Within an individual, NK cell diversity is also generated by the stochastic expression of KIR genes in some but not all NK cells. KIR alleles have not been previously described in pig-tailed macaques, but human KIR3DL alleles have been linked to slower disease progression in HIV-infected individuals. We cloned 23 different KIR alleles from 4 selected animals. The majority of these alleles encoded molecules with three immunoglobulin (Ig)-like domains and long or short cytoplasmic tail (KIR3DL or KIRDS). KIR alleles with one Ig-like domain (KIR1D) or lacking such domain (KIR0D) were also identified in each animal. We expressed 8 KIR alleles (4KIR3DL and 4 KIR3DS) in the MHC deficient 721.221 cell line and tested their ability to bind the three tetramers. One KIR3DL allele (KIR049-4) interacted strongly with A1*082 tetramers but not with A4*1402 tetramer, corresponding to type I receptor properties. The seven other KIR3D alleles did not recognize any tetramer. Incubation of primary macaque NK cells with 721.221 cells expressing A1*082 resulted in actin-dependent internalization of KIR049-4 and inhibition of NK cell activation / degranulation. A similar observation was obtained after stimulation with A1*084-expressing cells. In contrast, NK cell stimulation with 721.221 cells lacking MHC expression or expressing different MHC-A and B alleles resulted in high levels of activation and degranulation. Based on MHC allele sequence comparison, the KIR049-4 binding site on the MHC molecule appears to map to the alpha 1 helix between amino acids 70 and 83. These findings indicate that KIR049-4 + NK cell activation is regulated by missing-self and could contribute to antiviral immunity, by lysing cells based on SIV down regulation of MHC class I molecules. Our study is the first to characterize the interaction between MHC alleles and a KIR3DL receptor in macaques and represents a major step forward in elucidating the role of NK cells in the SIV / macaque model of infection.